Haemophilus influenzae is a human-restricted bacterium which can exist as an upper respiratory commensal, but also cause focal and/or systemic disease. More than 95 percent of the nasopharyngeal isolates lack capsules and are not serotypeable, and cause focal infections, while encapsulated (and rare nontypeable) strains cause sepsis and meningitis. The genome of both commensal and pathogenic isolates is 250 kb (on average) larger than the avirulent laboratory strain (Rd KW2O) whose genome sequence was published in 1995. We have found that the genome of strain Rd KW2O is a "scaffold" on which genes, gene clusters and operons encoding virulence factors are inserted, usually at the Haemophilus-specific DNA uptake sites. Using differential hybridization we will identify those DNA loci that permit nasopharyngeal colonization by commensals, and the additional putative virulence loci in pathogenic isolates. Each of these DNA fragments and the flanking DNA, will be sequenced until the Rd KW2O scaffold is identified. All sequence information will be posted on the UW Genome Center web site. Using strict criteria for homology (not conventional criteria) putative virulence genes will either be deleted or mutated and the virulence of the mutant compared to the parent in a relevant in vitro and/or in vivo infection model. For example a putative adhesin present in a lower respiratory tract isolate could be tested with human respiratory epithelial cell lines, and with primary human respiratory epithelium growing at an air-liquid interface in tissue culture. One unique isolate, an untypeable strain causing meningitis in an immunocompetent child immunized with a Hib-conjugate vaccine appears to be a member of a clade; we wish to confirm the preliminary finding and initiate a global database (in collaboration with the MLST Haemophilus Center) for these vaccine-failure isolates. With an understanding of the molecular mechanisms of colonization versus infection, strategies for prevention and intervention can be devised.